1. Technical Field
The subject invention relates to a novel hepatitis B surface antigen mutant and methods of detecting this mutant, and/or antibodies thereto, in patient samples. In particular, the mutant contains a substitution of amino acid threonine for the amino acid alanine at position 123 in the amino acid sequence of the hepatitis B surface antigen (HBsAg) protein.
2. Background Information
The hepatitis B virus (HBV) is known to cause a variety of disease states from mild subclinical infection to chronic active and fulminant hepatitis. The genome of the virus is a circular, partially double stranded DNA sequence of approximately 3200 basepairs which code for at least six different viral genes (Tiollais et al., Nature 317:489–495 (1985)). More specifically, the polymerase gene overlaps the envelope gene and also partially overlaps the X and core genes. The product of the envelope gene consists of three proteins which have different initiation sites but the same termination site. These three proteins (i.e., small (S), middle (M), and large (L) HBsAg) all contain the S-HBsAg gene sequence of 226 amino acids (Gerlich et al. in Viral Hepatitis and Liver Disease, Hollinger et al., eds., Williams-Wilkens, Baltimore, Md., pages 121–134 (1991)). The M-HBsAg contains the 55 amino acid PreS2 sequence and the S sequence for a total length of 281 amino acids. The L-HBsAg protein contains the 108 amino acid PreS1 sequence plus the PreS2 and S sequences for a total length of 389 amino acids. In addition, each of the three envelope proteins exhibit different degrees of glycosylation.
The core gene encodes the nucleocapsid protein, hepatitis B core antigen (HBcAg). Immediately upstream of the core gene is the precore region. The first 19 amino acids of the precore region serve as a signal for membrane translocation and eventual secretion of the precore gene product, the hepatitis B e antigen (HBeAg).
Similar to the Human Immunodeficiency Virus (HIV), HBV uses reverse transcriptase (RT) as an essential step in the replication cycles. However, RT has poor proofreading ability, thereby leading to a high rate of nucleotide misincorporation. Calculations suggest that this error-prone replication leads to one point replacement, deletion or insertion per 1000 to 100,000 nucleotides copied (Carman et al., Lancet 341:349–353 (1993)). Variability in HBV surface antigen was first described using classical subtyping studies Courouce et al., Bibliotheca Haematologica 42:1 (1976)).
The HBV envelope regions encompassing PreS1 and PreS2 and the “a” determinant are exposed on the surface of the viral particle and are therefore expected to be targets of immune surveillance (Gerlich et al., supra). Some surface antigen mutants previously described have significantly affected the antigenicity of the “a” determinant which contains both common and group-specific determinants (Carman et al., Gastroenterology 102:711–719 (1992)). The “a” determinant is located between amino acids 100–160 of S-HBsAg and presents a complex conformational epitope which is stabilized by disulfide bonding between highly conserved cysteine residues. The “a” determinant immunoreactivity can be partially mimicked using cyclic synthetic peptides. Further, although the “a” determinant had been traditionally defined by reactivity to polyclonal antisera, the use of monoclonal antibody has shown that the “a” determinant consists of at least five partially overlapping epitopes (Peterson et al., J. Immunol. 132:920–927 (1984)). The most common surface antigen mutant described in the literature is a single nucloetide substitution leading to the substitution of glycine at amino acid position 145 of S-HBsAg with arginine (G-R 145). This G-R 145 mutation destroys some, but not all, “a” determinant epitopes.
Additionally, other mutations in the “a” determinant result in loss of subtypic or type-specific determinants y/d and w/r. Also the emergence of gross deletions and point mutations in the PreS1/PreS2 region suggest that the product of the envelope gene is under immune selection in chronically infected patients. Further, HBV mutants which cannot replicate because of deletions in the env, C or P genes have been noted in plasma from HBV carriers. All co-exist with HBV forms which are replication competent.
Okamoto et al. have demonstrated that mutant genomes with gross deletions in the PreS/S, C and P genes derived from plasma or asymptomatic carriers may be complemented in transient expression systems with hepatoma cells (Okamoto et al., Pediatric Research 32:264–268 (1992)). In fact, the suggestion has been made that HBV mutants acting as defective interfering particles may attenuate wildtype virus replication and thereby help maintain persistence of the invention.
In view of the above, the isolation of Hepatitis B surface antigen mutants is certainly advantageous. Furthermore, new mutants may arise over time due to vaccine administration and/or infection. The identification and detection of mutant Hepatitis B viruses may thus lead to improved vaccine development and to detection systems which determine the presence of these mutants in patient samples.
All U.S. patents and publications are herein incorporated in their entirety by reference.